Device and method for multiple analyte detection

ABSTRACT

The invention is directed to a method and device for simultaneously testing a sample for the presence, absence, and/or amounts of one or more of a plurality of selected analytes. The invention includes, in one aspect, a device for detecting or quantitating a plurality of different analytes in a liquid sample. Each chamber may include an analyte-specific reagent effective to react with a selected analyte that may be present in the sample, and detection means for detecting the signal. Also disclosed are methods utilizing the device.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/029,968 filed Jan. 5, 2005, which is a continuation application ofU.S. patent application Ser. No. 09/628,076, filed Jul. 28, 2000, whichis a continuation application of U.S. patent application Ser. No.08/831,983, filed Apr. 2, 1997, now U.S. Pat. No. 6,126,899, whichclaims the benefit of prior Provisional Application No. 60/014,712,filed Apr. 3, 1996, all of which are incorporated herein in theirentireties by reference.

FIELD OF THE INVENTION

The present invention relates to devices and methods for detecting orquantifying one or more selected analytes in a sample.

REFERENCES

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BACKGROUND OF THE INVENTION

Biochemical testing is becoming an increasingly important tool fordetecting and monitoring diseases. While tests have long been known forobtaining basic medical information such as blood type and transplantcompatibility, for example, advances in understanding the biochemistryunderlying many diseases have vastly expanded the number of tests whichcan be performed. Thus, many tests have become available for variousanalytical purposes, such as detecting pathogens, diagnosing andmonitoring disease, detecting and monitoring changes in health, andmonitoring drug therapy.

An important obstacle which has limited exploitation of many biochemicaltests has been cost. Simultaneous testing of multiple samples for asingle analyte has provided some savings. However, simultaneous assaysfor a large number of analytes within a single sample have been lesspractical because of the need for extended sample manipulation, multipletest devices, multiple analytical instruments, and other drawbacks.

Ideally, a method for analyzing an individual sample using a single testdevice should provide diagnostic information for a large number ofpotential analytes while requiring a small amount of sample. The deviceshould be small in size while providing high-sensitivity detection forthe analytes of interest. The method should also require minimal samplemanipulation. Preferably, the device will include pre-dispensed reagentsfor specific detection of the analytes.

SUMMARY OF THE INVENTION

The present invention is directed generally to a method and device forsimultaneously testing a sample for the presence, absence and/or amountof one or more selected analytes.

The invention includes, in one aspect, a device for detecting orquantitating one or more of a plurality of different analytes in aliquid sample. The device includes a substrate which defines asample-distribution network having (i) a sample inlet, (ii) one or moredetection chambers, and (iii) channel means providing a dead-end fluidconnection between each of the chambers and the inlet. Preferably, eachchamber includes an analyte-specific reagent effective to react with aselected analyte that may be present in the sample, and detection meansfor detecting the signal.

In one embodiment, the detection means for each chamber includes anoptically transparent window through which the signal can be detectedoptically. In another embodiment, the detection means includes anon-optical sensor for detecting the signal.

The channel means of the device may be configured in numerous ways. Forexample, in one embodiment, the channel means includes a single channelto which the detection chambers are connected by dead-end fluidconnections. In a second embodiment, the channel means includes at leasttwo different channels, each connected to a different group of detectionchambers. In yet another embodiment, the channel means includes anindividual channel for each detection chamber.

The device may include a vacuum port for placing the detection chambersunder vacuum prior to the addition of sample. In one embodiment, thevacuum port is connected to the channel means at a site between, and influid communication with, the sample inlet and the detection chambers.In another embodiment, the vacuum port is connected to the channel meansat a site downstream of the detection chambers. In this configuration,the vacuum port is additionally useful for removing liquid from thechannel means after the detection chambers have been filled, to helpisolate the detection chambers from one another and further reducecross-contamination.

The vacuum port may be incorporated in a multi-port valve (e.g., a 3-wayvalve) that permits the network and associated detection chambers to beexposed alternately to a vacuum source, the sample inlet, and a vent orselected gas source.

Alternatively, the device of the invention is prepared and sealed undervacuum when manufactured, so that a vacuum port is unnecessary.

According to an important feature of the invention, the device iscapable of maintaining a vacuum within the sample-distribution network(low internal gas pressure, relative to the external, ambient pressureoutside the device) for a time sufficient to allow a sample to be drawninto the network and distributed to the detection chambers by vacuumaction. For this purpose, the sample-distribution network may include avacuum reservoir in fluid communication with, and downstream of, thedetection chambers, for preventing the build-up of back-pressure in thenetwork while the detection chambers are successively filled.

In one embodiment, the vacuum reservoir includes a non-flowthroughcavity connected downstream of the last-filled detection chamber, foraccumulating residual gas displaced from the inlet and channel means. Inanother embodiment, the reservoir comprises the terminal end of thechannel means connected to a vacuum source, allowing residual gasdisplaced by the sample to be removed continuously until sample loadingis complete.

The analyte-specific reagents in the detection chambers may be adaptedto detect a wide variety of analyte classes, including polynucleotides,polypeptides, polysaccharides, and small molecule analytes, for example.In one embodiment, the analytes are selected-sequence polynucleotides,and the analyte-specific reagents include sequence-selective reagentsfor detecting the polynucleotides. The polynucleotide analytes aredetected by any suitable method, such as polymerase chain reaction,ligase chain reaction, oligonucleotide ligation assay, or hybridizationassay.

In one particular embodiment, for polynucleotide detection, theanalyte-specific reagents include an oligonucleotide primer pairsuitable for amplifying, by polymerase chain reaction, a targetpolynucleotide region in the selected analyte which is flanked bysequences complementary to the primer pair. The presence of targetpolynucleotide, as indicated by successful amplification, is detected byany suitable means.

In another embodiment, the analyte-specific reagents in each detectionchamber include an antibody specific for a selected analyte-antigen. Ina related embodiment, when the analyte is an antibody, theanalyte-specific detection reagents include an antiben for reacting witha selected analyte antibody which may be present in the sample.

In yet another embodiment, the device includes means for regulating thetemperatures of the detection chambers, preferably providing temperaturecontrol between 0 EC and 100 EC, for promoting the reaction of thesample with the detection reagents. In one preferred embodiment, thetemperature regulating means includes a conductive heating element foreach detection chamber, for rapidly heating the contents of the chamberto a selected temperature. The temperature control means is preferablyadapted to regulate the temperatures of the detection chambers, forheating and cooling the chambers in accordance with a selected assayprotocol.

The device may be manufactured and packaged so that thesample-distribution network (e.g., sample inlet, detection chambers, andchannel means) is provided under vacuum, ready for immediate use by theuser. Alternatively, the sample-distribution network is provided underatmospheric pressure, so that the evacuation step is carried out by theend-user prior to sample loading.

The invention also includes a substrate containing a plurality ofsample-distribution networks as described above, for testing a singlesample or a plurality of samples for selected analytes.

In another aspect, the invention includes a method of making a devicesuch as described above.

In another aspect, the invention includes a method for detecting orquantitating a plurality of analytes in a liquid sample. In the method,there is provided a device of the type described above, wherein theinterior of the network is placed under vacuum. A liquid sample is thenapplied to the inlet, and the sample is allowed to be drawn into thesample-distribution network by vacuum action, delivering sample to thedetection chambers. The delivered sample is allowed to react with theanalyte-specific reagent in each detection chamber under conditionseffective to produce a detectable signal when the selected analyte ispresent in the sample. The reaction chambers are inspected or analyzedto determine the presence and/or amount of the selected analytes in thesample.

The device of the invention may also be provided as part of a kit whichadditionally includes selected reagents, sample preparation materials ifappropriate, and instructions for using the device.

These and other objects and features of the invention will be moreapparent from the following detailed description when read in light withthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a plan view (1A) and perspective view (1B) of anexemplary assay device in accordance with the invention;

FIGS. 2A-2C illustrate several exemplary sample distribution networkconfigurations in accordance with the invention;

FIGS. 3A-3C illustrates a time sequence for the filling of the detectionchambers of a sample-distribution network with fluid sample;

FIG. 4 illustrates a sample-distribution network containing three sampledelivery channels for delivering sample to three different sets ofdetection chambers;

FIG. 5 illustrates a sample-distribution network having a separatedelivery channel for each detection chamber;

FIGS. 6A-6C illustrate selected features of another sample-distributionnetwork in accordance with the invention; the device is shown in planview (6A), perspective view (6B), with a portion of the sampledistribution network of the device shown in FIG. 6C;

FIG. 7 shows an exploded view of a portion of a device in accordancewith the invention;

FIG. 8 shows an exploded view of a portion of another device inaccordance with the invention; and

FIG. 9 shows a perspective view of another device in accordance with theinvention.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The following terms and phrases as used herein are intended to have themeanings below.

“Dead-end fluid connection between a detection chamber and a sampleinlet” refers to a fluid connection which provides the sole fluid accessto a detection chamber, such that fluid cannot enter or exit thedetection chamber by any other way than through the dead-end fluidconnection.

In particular, “dead-end fluid connection” refers to a channel whosecross-section is sufficiently narrow to preclude bi-directional fluidflow through the channel. That is, liquid cannot flow through thechannel in one direction while air or another liquid is flowing throughthe channel in the opposite direction.

As used herein, “microdevice” means a device in accordance with theinvention.

II. Assay Device

In one aspect, the present invention provides a device which is usefulfor testing one or more fluid samples for the presence, absence, and/oramount of one or more selected analytes. The device includes a substratewhich defines a sample-distribution network having (i) a sample inlet,(ii) one or more detection chambers (preferably a plurality of detectionchambers), and (iii) channel means providing a dead-end fluid connectionbetween each of the chambers and the inlet. Each chamber includes ananalyte-specific reagent effective to react with a selected analyte thatmay be present in such sample.

In one embodiment, the substrate also provides, for each chamber, anoptically transparent window through which analyte-specific reactionproducts can be detected. In another embodiment, the detection means foreach chamber comprises a non-optical sensor for signal detection.

The present invention provides a number of advantages in an assay formultiple analytes in a sample, as will be discussed below. Inparticular, the invention facilitates the transition from a macro sizesample to a micro-sized sample, wherein the device of the inventionprovides one-step metering of reagents and sample in a multi-analytedetection assay.

A. Network Configurations

FIGS. 1A and 1B show a plan view and perspective view, respectively, ofan exemplary assay device 30 in accordance with the invention. Device 30includes a substrate 32 which defines a sample-distribution network 34.With reference to FIG. 1B, the device also includes mount 36 containinga sample inlet 38 and optionally, vacuum port means 40 which is locateddownstream of the detection chambers.

Inlet 38 may be adapted to form a vacuum-tight seal with the end of asyringe, for sample loading, or with a multi-port valve to provide fluidcommunication with the sample and one or more liquid or gaseous fluids.The inlet may further include a septum cap, if desired, for maintainingthe network under vacuum and allowing introduction of sample by canulaor needle.

Vacuum port 40 may be adapted for connection to a vacuum source, such asa vacuum pump. The vacuum connection may include a valve for closing offthe sample-distribution network from the vacuum source, or a multi-portvalve for connection to a vacuum source and one or more selected gassupplies.

Substrate 30 further provides indentations or holes 42, which may bearranged asymmetrically as illustrated in FIG. 1A, to engagecorresponding pins or protrusions in a device-holder, not shown, toimmobilize and orient the device for analysis.

As noted in the Summary of the Invention, the sample-distributionnetwork of the invention may utilize any of a number of differentchannel configurations, or channel means, for delivering sample to theindividual detection chambers. With reference to FIG. 2A, distributionnetwork 34 a includes sample inlet 38 a, a plurality of detectionchambers 44 a, channel means comprising a single channel 46 a to whichthe detection chambers are each connected by dead-end fluid connections48 a, and a vacuum port 40 a. The detection chambers are distributed oneither side of channel 46 a, with the fluid connections branching off inpairs from opposite sides of the channel. FIG. 2B shows a portion of analternative network 34 b having an inlet 38 b and detection chambers44B, wherein fluid connections 48 b branch off from channel 46 b in astaggered manner.

The detection chambers in the device of the invention may be arranged toform a repeating 2-dimensional array which facilitates indexing andidentification of the various chambers, as well as allowing rapidmeasurement of an optical signal produced by each chamber upon reactionwith the sample, if optical detection is used.

FIGS. 2A-2B, for example, show networks in which the detection chambersare arranged in rows and columns along perpendicular axes, allowing thechambers to be identified by X and Y indices if desired. This type ofarray (a perpendicular array) also facilitates successive interrogationsof the chambers in a chamber-by-chamber analysis mode. However, otherarrangements may be used, such as a staggered or a close-packedhexagonal array. FIG. 2C, for example, shows part of a network 34 chaving inlet 38 c and an array of staggered detection chambers 44 c. Thedetection chambers are connected to a common delivery channel 46 c byfluid connections 48 c.

The device may also include identifying symbols adjacent the detectionchamber to facilitate identification or confirmation of the analytesbeing detected.

Preferably, the detection chambers of the device are each provided withanalyte-specific reagents which are effective to react with a selectedanalyte which may be present in the sample, as discussed further below.Reaction of the sample with the analyte-specific reagents results inproduction of a detectable signal which indicates that the selectedanalyte is present.

According to an important feature of the invention, the sample isdelivered to the detection chambers by vacuum action. Prior to loadingwith sample, the interior of the sample-distribution network is placedunder vacuum so that the residual gas pressure in the network issubstantially below atmospheric pressure (i.e., substantially less than760 mm Hg). One advantage of this feature of the invention is that apump for pushing fluid through the network is not required. Instead, thedevice exploits ambient atmospheric pressure to push the sample throughthe sample inlet and into the sample-distribution network. This allowsthe sample to be delivered quickly and efficiently to the detectionchambers.

FIGS. 3A-3C illustrate the filling process for a sample-distributionnetwork 34 in accordance with FIG. 2A. The network includes sample inlet38, detection chambers 44, and sample delivery channel 46 which isconnected to the various detection chambers by dead-end fluidconnections 48. The network further includes a vacuum reservoir 40 atthe terminus of the delivery channel. A plurality of the detectionchambers 44 contain dried detection reagents for detecting a differentselected analyte in each chamber, with one or more chambers optionallybeing reserved as controls.

FIG. 3A shows the device before sample loading is initiated. The networkis evacuated to establish an internal pressure within the network thatis substantially below atmospheric pressure (e.g., 1 to 40 mm Hg). Theinterior of the network should also be substantially liquid-free tominimize vapor pressure problems. FIG. 3B shows the network after samplefluid 50 has entered the network through inlet 38 (FIG. 3B). As thesample moves through channel 46, the sample sequentially fills each ofthe detection chambers (FIG. 3B) until all of the chambers have beenfilled (FIG. 3C). With continued reference to FIG. 3C, once thedetection chambers have all been filled, sample fluid may continue toflow through channel 46 into vacuum reservoir 40 until the reservoirbecomes full or the flow is otherwise terminated (e.g., by closing avalve associated with the vacuum reservoir).

According to one advantage of the invention, continued sample flowthrough the channel means does not substantially disturb the contents ofthe detection chambers that have already been filled, because furtherflow into or out of each filled detection chamber is restricted by thedead-end fluid connections, such as connections 48. Cross-contaminationbetween different detection chambers is therefore reduced, so thaterroneous signals due to cross-contamination can be avoided. A furtheradvantage of the invention is that the sample can be mixed with theanalyte-specific detection reagents and detected all in the samechamber, without requiring movement of the sample from each chamber toanother site. Moreover, since the sample and detection reagents canremain in the chamber for signal detection, the detection reagents neednot be immobilized on or adhered to the inner surfaces of the detectionchambers.

The components of the sample-distribution network are designed to ensurethat an adequate volume of sample will be delivered to the detectionchambers to allow accurate analyte detection and/or quantitation. Ingeneral, the percent-volume of a detection chamber that must be occupiedby the sample will vary according to the requirements of the reagentsand the detection system used. Typically, the volume-percent will begreater than 75%, preferably greater than 90%, and more preferablygreater than 95%. In assay formats in which the detection chambers areheated, particularly to temperatures of between about 60 EC and about 95EC, the volume-percent filling of the chambers is preferably greaterthan 95%, and more preferably is at least 99%.

The degree to which the detection chambers are filled with sample willgenerally depend upon (1) the initial ratio of the external(atmospheric) pressure to the initial pressure within the network, (2)the individual and total volumes defined by the detection chambers, (3)the volume defined by the channel means, and (4) the nature of thenetwork downstream of the last detection chamber.

For example, in the case of a detection chamber which is nearest thesample inlet, and which will be filled first, the percentage occupancy(volume-percent) of sample fluid in the chamber after sample loading(V_(s,%)) will be related to the external atmospheric pressure (P_(ext))and the initial internal pressure within the network before sampleloading (P_(int)) by the expression:V_(s,%)·(P_(ext))/(P_(ext)+P_(int))

Thus, if the initial pressure within the network (P_(int)) is 10 mm Hg,and the external pressure (P_(ext)) is 760 mm Hg, about 99% of the firstdetection chamber will be filled with sample fluid (V_(s,%)·99%), withthe remaining volume (. 1.3%) being filled by residual gas (e.g., air)displaced by the sample. (This calculation assumes that, by the time thesample reaches the chamber, the internal network pressure has notincreased appreciably due to displacement of gas upstream of thechamber.) Similarly, if P_(ext) is 760 mm Hg and P_(int) is only 40 mmHg, the volume-percent of the chamber that becomes occupied with samplewill still be very high (about 95%).

It will be appreciated that as the sample fluid reaches and fillssuccessive detection chambers, the residual gas displaced from thechannel means will gradually accumulate in the remaining network volume,so that the internal pressure will gradually increase. The resultantincrease in back-pressure can lead to a reduction in V_(s,%) for eachsuccessive chamber, with V_(s,%) for the last-filled detection chamberbeing significantly lower than the V_(s,%) for the first-filled chamber.

To help avoid this problem, the dimensions of the channel and dead-endfluid connections are preferably selected to define a total volume thatis substantially less than the total volume defined by the detectionchambers. Preferably, the collective volume of the channel means is lessthan 20% of the total collective volume of the detection chambers, andmore preferably less than 5%. Similarly, the volume of each dead-endfluid connection should be substantially less than the volume of theassociated detection chamber. Preferably, the volume of each dead-endconnection is less than 20%, preferably less than 10%, and morepreferably less than 5% of the volume of the associated detectionchamber.

The problem of back-pressure can be further diminished by including avacuum reservoir downstream of the last detection chamber to be filled.In one embodiment, the vacuum reservoir is a non-flowthrough cavity inwhich gas displaced by the sample fluid can collect. The volume of thereservoir will vary according to the configuration and needs of theparticular device. For example, the volume of the reservoir can beselected to be equal in volume to one or more detection chambersvolumes, or alternatively, is one- to five-fold as great as the totalcollective volume of the channel means.

In another embodiment, the vacuum reservoir is connected to a vacuumsource, so that residual gas can be removed continuously until sampleloading into the detection chambers is complete, as discussed furtherbelow.

FIG. 4 shows another sample-distribution network in accordance with theinvention, wherein the channel means of the network includes at leasttwo different sample delivery channels, each connected to a differentgroup of detection chambers. FIG. 4 shows a sample-distribution network60 having a sample inlet 62, three different groups of detectionchambers 64 a, 64 b, and 64 c, and channel means 66 which includecorresponding channels 66 a, 66 b, and 66 c associated with the threechamber groups. The chambers are connected to channels 64 a-64 c viadead-end fluid connections 68 a-68 c, which provide unidirectional flowof the sample into the detection chambers.

One advantage of using multiple delivery channels is that the timeneeded to fill the detection chambers with the sample can besignificantly reduced relative to the time needed to fill the samenumber of detection chambers using a single delivery channel. Forexample, the loading time for a network in accordance with FIG. 4 willbe about one-third of that needed to fill an identical number ofdetection chambers via the single channel format illustrated in FIG. 2A,all other factors being equal. More generally, for a given number ofdetection chambers, the filling time will vary inversely with the numberof delivery channels used.

The sample-distribution network in FIG. 4 further includes separatevacuum reservoirs 70 a-70 c which are connected to the termini of sampledelivery channels 66 a-66 c, downstream of the detection chambers. Thevacuum chambers are dimensioned to help maintain a low internal gaspressure during sample loading.

In another embodiment, the channel means includes an individual channelfor each detection chamber, as illustrated in FIG. 5. Network 80includes an inlet 82, detection chambers 84, and associated with eachdetection chamber, a dead-end fluid connection 86, which may also bereferred to as channel means, for delivering sample to each chamber.Each dead-end fluid connection is dimensioned to define a volume that issubstantially less than the volume of the associated detection chamber,to ensure that each detection chamber is sufficiently filled withsample. This embodiment provides rapid filling of the detection chamberswith minimal cross-contamination.

The device of the invention may also include a vacuum port communicatingwith the sample-distribution network, for applying a vacuum to thenetwork before or during sample loading. In one embodiment, the vacuumport is connected to the channel means at a site between, and in fluidcommunication with, the sample inlet and the detection chambers. Anillustration of this can be found in FIG. 9. The vacuum port thusprovides a convenient way to reduce the internal pressure within thenetwork to a selected residual pressure prior to sample loading. Inparticular, when the sample is introduced into the network using asyringe barrel connected to the sample inlet, the vacuum port can beused to remove air from the space between the syringe and the inlet,before the sample is admitted into the network.

In another embodiment, the vacuum port is connected to the channel meansat a site downstream of the sample inlet and detection chambers (e.g.,FIG. 6A). In this configuration, the vacuum port may additionally beused to remove liquid from the channel means after the detectionchambers have been filled, to help isolate the detection chambers fromone another and further reduce cross-contamination. In thisconfiguration, the vacuum port constitutes a part of the vacuumreservoir described above, where the reservoir includes a vacuum sourcelinked to the terminal end of a sample delivery channel. The vacuum portmay be kept open to the network during sample loading, to continuouslyremove residual gas from the network until all of the detection chambershave been filled.

The vacuum port may include a multi-port valve (e.g., 3-way valve) thatpermits the network and associated detection chambers to be exposedalternately to a vacuum source, the sample inlet, and a vent or gassource. Such a valve may be used to alternately expose the network tovacuum and a selected gas source, to replace residual air with theselected gas. Such gas replacement in the network may be useful toremove molecular oxygen (O₂) or other atmospheric gases which mightotherwise interfere with the performance of the detection reagents.

Argon and nitrogen are inert gases which may be suitable for mostsituations. Another gas which may be used is carbon dioxide (CO₂), whichis highly soluble in water due to its ability to form carbonate andbicarbonate ions. When the sample fluid is an aqueous solution, bubblesof carbon dioxide which may form in the network during sample loadingmay be eliminated via dissolution in the sample fluid. The degree ofsample filling in the detection chambers is therefore enhanced. Ofcourse, carbon dioxide should not be used if it interferes with thedetection reagents.

A multi-port valve such as noted above can also be used to supply a gaswhich is required for detection of the selected analytes. For example,it may be desirable to provide molecular oxygen or ozone where thedetection reagents involve an oxidation reaction. Other gases, such ashydrocarbons (ethylene, methane) or nitrogenous gases, may also beintroduced as appropriate.

B. Device Fabrication

The device of the invention is designed to allow testing of a sample fora large number of different analytes by optical analysis, using a devicethat is compact and inexpensive to prepare. Generally, the device willbe no larger in cross-section than the cross-section of a standardcredit card (<5 cm×10 cm), and will have a thickness (depth) of nogreater than 2 cm. More preferably, the device occupies a volume of nogreater than about 5×5×1 cm, excluding attachments for the sample inletand any vacuum port. More preferably, the device has dimensions of nogreater than about 3 cm×2 cm×0.3 cm. Devices smaller than this are alsocontemplated, bearing in mind that the device should provide adequatesensitivity and be easy for the end-user to handle.

The detection chambers in the device are generally designed to be assmall as possible, in order to achieve high density of detectionchambers. The sizes and dimensions of the chambers will depend on anumber of considerations. When signal detection is by optical means, theoverhead cross-section of each chamber must be large enough to allowreliable measurement of the signal produced when the selected analyte ispresent in the sample. Also, the depths of the chambers can be tailoredfor the particular optical method used. For fluorescence detection, thinchambers may be desirable, to minimize quenching effects. For absorbanceor chemiluminescence detection, on the other hand, a thicker chamber maybe appropriate, to increase the detection signal.

It will be appreciated that while the figures in the attached drawingsshow chambers having square-shaped overhead cross-sections, othergeometries, such as circles or ovals, may also be used. Similarly, thechannels in the network may be straight or curved, as necessary, withcross-sections that are shallow, deep, square, rectangular, concave, orV-shaped, or any other appropriate configuration.

Typically, the detection chambers will be dimensioned to hold from 0.001μL to 10 μL of sample per chamber, and, more preferably between 0.01 μLand 2 μL. Conveniently, the volume of each detection chamber is betweenabout 0.1 μL and 1 μL, to allow visual confirmation that the chambershave been filled. For example, a chamber having a volume of 0.2 μL mayhave dimensions of 1 mm×1 mm×0.2 mm, where the last dimension is thechamber's depth.

The sample delivery channels are dimensioned to facilitate rapiddelivery of sample to the detection chambers, while occupying as littlevolume as possible. Typical cross-sectional dimensions for the channelswill range from 5 μm to about 250 μm for both the width and depth.Ideally, the path lengths between chambers will be as short as possibleto minimize the total channel volume. For this purpose (to minimizevolume), the network is preferably substantially planar, i.e., thechannel means and detection chambers in the device intersect a commonplane.

The substrate that defines the sample-distribution network of theinvention may be formed from any solid material that is suitable forconducting analyte detection. Materials which may be used will includevarious plastic polymers and copolymers, such as polypropylenes,polystyrenes, polyimides, and polycarbonates. Inorganic materials suchas glass and silicon are also useful. Silicon is especially advantageousin view of its high thermal conductivity, which facilitates rapidheating and cooling of the device if necessary. The substrate may beformed from a single material or from a plurality of materials.

The sample-distribution network is formed by any suitable method knownin the art. For plastic materials, injection molding will generally besuitable to form detection chambers and connecting channels having adesired pattern. For silicon, standard etching techniques from thesemiconductor industry may be used, as described in Sze (1988), forexample.

Typically, the device substrate is prepared from two or more laminatedlayers, as will be discussed below with reference to FIGS. 6A-6C to 8.For optical detection, the device will include one or more layers whichprovide an optically transparent window for each detection chamber,through which the analyte-specific signal is detected. For this purpose,silica-based glasses, quartz, polycarbonate, or an optically transparentplastic layer may be used, for example. Selection of the particularwindow material depends in part on the optical properties of thematerial. For example, in a fluorescence-based assay, the materialshould have low fluorescence emission at the wavelength(s) beingmeasured. The window material should also exhibit minimal lightabsorption for the signal wavelengths of interest.

Other layers in the device may be formed using the same or differentmaterials. Preferably, the layer or layers defining the detectionchambers are formed predominantly from a material that has high heatconductivity, such as silicon or a heat-conducting metal. The siliconsurfaces which contact the sample are preferably coated with anoxidation layer or other suitable coating, to render the surface moreinert. Similarly, where a heat-conducting metal is used in thesubstrate, the metal can be coated with an inert material, such as aplastic polymer, to prevent corrosion of the metal and to separate themetal surface from contact with the sample. The suitability of aparticular surface should be verified for the selected assay.

For optical detection, the opacity or transparency of the substratematerial defining the detection chambers will generally have an effecton the permissible detector geometries used for signal detection. Forthe following discussion, references to the “upper wall” of a detectionchamber refer to the chamber surface or wall through which the opticalsignal is detected, and references to the “lower wall” of a chamberrefers to the chamber surface or wall that is opposite the upper wall.

When the substrate material defining the lower wall and sides of thedetection chambers is optically opaque, and detection is by absorptionor fluorescence, the detection chambers will usually be illuminated andoptically scanned through the same surface (i.e., the top surfaces ofthe chambers which are optically transparent). Thus, for fluorescencedetection, the opaque substrate material preferably exhibits lowreflectance properties so that reflection of the illuminating light backtoward the detector is minimized. Conversely, a high reflectance will bedesirable for detection based on light absorption.

When the substrate material defining the upper surface and sides of thedetection chambers is optically clear, and detection involvesfluorescence measurement, the chambers can be illuminated withexcitation light through the sides of the chambers (in the plane definedcollectively by the detection chambers in the device), or moretypically, diagonally from above (e.g., at a 45 degree angle), andemitted light is collected from above the chambers (i.e., through theupper walls, in a direction perpendicular to the plane defined by thedetection chambers). Preferably, the substrate material exhibits lowdispersion of the illuminating light in order to limit Rayleighscattering.

When the entirety of the substrate material is optically clear, or atleast the upper and lower walls of the chambers are optically clear, thechambers can be illuminated through either wall (upper or lower), andthe emitted or transmitted light is measured through either wall asappropriate. Illumination of the chambers from other directions willalso be possible as already discussed above.

With chemiluminescence detection, where light of a distinctivewavelength is typically generated without illumination of the sample byan outside light source, the absorptive and reflective properties of thesubstrate will be less important, provided that the substrate providesat least one optically transparent window for detecting the signal.

FIGS. 6A-6C illustrate a specific embodiment of a device in accordancewith the invention. With reference to FIGS. 6A and 6B, device 100includes a sample inlet 102, sample-distribution network 104, and vacuumport 106 which is connected to the terminus of network 104. Network 104includes a perpendicular array of detection chambers 108 (7 rows×8columns) linked to sample delivery channel 110 via dead-end fluidconnections 112. The device further includes vertical panel 114 adjacentsample inlet 102, for attaching an identifying label to the device andas an attachment allowing the user to hold the device.

As can be seen from FIG. 6B, the detection chambers are packed closelytogether to increase the number of analytes which can be tested in thedevice. Fluid connections 112 are provided in an L-shaped configuration(FIG. 6C) to impede fluid flow out of the chambers after sample loading,and to help isolate the contents of the chambers from each other.Although the horizontal rows of detection chambers in FIGS. 6A and 6Bare shown as being separated from each other by variable verticalspacing (to enhance the clarity of the figures), it will be appreciatedthat the chambers can be separated by equal distances in both thevertical and horizontal directions, to facilitate analysis of thechambers.

FIGS. 7 and 8 illustrate two exemplary approaches for forming a testingdevice in accordance with FIGS. 6A-6B. FIG. 7 shows two substrate layers140 and 142 which can be brought together to form sample-distributionnetwork 104 (FIG. 6A). The network is defined primarily by substratelayer 140, which contains indentations defining a sample inlet 102 (notshown), a plurality of detection chambers 108, sample delivery channel110, and dead-end fluid connections 112. Contact of substrate layer 142with the opposing face of layer 140 completes the formation of network104.

FIG. 8 shows substrate layers 150 and 152 for forming a network byanother approach. Substrate layer 150 contains indentations defining aplurality of detection chambers 108. Substrate layer 152, on the otherhand, contains indentations defining sample delivery channel 110 anddead-end fluid connections 112. Network 104 can then be formed bycontacting the opposing faces of the two substrate layers as in FIG. 7.

Since the device is designed to provide a vacuum-tight environmentwithin the sample-distribution network for sample loading, and also toprovide detection chambers having carefully defined reaction volumes, itis desirable to ensure that the network and associate detection chambersdo not leak. Accordingly, lamination of substrate layers to one anothershould be accomplished so as to ensure that all chambers and channelsare well sealed.

In general, the substrate layers can be sealably bonded in a number ofways. Conventionally, a suitable bonding substance, such as a glue orepoxy-type resin, is applied to one or both opposing surfaces that willbe bonded together. The bonding substance may be applied to the entiretyof either surface, so that the bonding substance (after curing) willcome into contact with the detection chambers and the distributionnetwork. In this case, the bonding substance is selected to becompatible with the sample and detection reagents used in the assay.Alternatively, the bonding substance may be applied around thedistribution network and detection chambers so that contact with thesample will be minimal or avoided entirely. The bonding substance mayalso be provided as part of an adhesive-backed tape or membrane which isthen brought into contact with the opposing surface. In yet anotherapproach, the sealable bonding is accomplished using an adhesive gasketlayer which is placed between the two substrate layers. In any of theseapproaches, bonding may be accomplished by any suitable method,including pressure-sealing, ultrasonic welding, and heat curing, forexample.

The device of the invention may be adapted to allow rapid heating andcooling of the detection chambers to facilitate reaction of the samplewith the analyte-detection reagents. In one embodiment, the device isheated or cooled using an external temperature-controller. Thetemperature-controller is adapted to heat/cool one or more surfaces ofthe device, or may be adapted to selectively heat the detection chambersthemselves.

To facilitate heating or cooling with this embodiment, the substratematerial of the test device is preferably formed of a material which hashigh thermal conductivity, such as copper, aluminum, or silicon.Alternatively, a substrate layer such as layer 140 in FIG. 7 may beformed from a material having moderate or low thermal conductivity,while substrate layer 142 (FIG. 7) is provided as a thin layer, suchthat the temperature of the detection chambers can be convenientlycontrolled by heating or cooling the device through layer 142,regardless of the thermal conductivity of the material of layer 142. Inone preferred embodiment, layer 142 is provided in the form of anadhesive copper-backed tape.

In an alternative embodiment, means for modulating the temperature ofthe detection chambers is provided in the substrate of the deviceitself. For example, with reference to FIG. 7, substrate layer 142 mayinclude resistive traces which contact regions adjacent the reactionchambers, whereby passage of electrical current through the traces iseffective to heat or cool the chambers. This approach is particularlysuitable for silicon-based substrates, and can provide superiortemperature control.

Further illustration of the invention is provided by the device shown inFIG. 9. Device 160 includes a network-defining substrate layer 161 and aflat substrate layer 180 for bonding with and sealing layer 161.

Layer 161 includes sample inlet 162 and indentations defining (i) asample-distribution network 164 and (ii) vacuum reservoir 166 connectedto the terminus of network 164. Network 164 includes a 2-dimensionalperpendicular array of detection chambers 168 (7×7) linked to sampledelivery channel 170 via dead-end fluid connections 172. The devicefurther includes vertical panel 174 adjacent sample inlet 162, as inFIGS. 6A-6B. Formation of network 164 is completed by contacting theentirety of the upper surface of device 160 with the opposing face of alayer 180, which is preferably provided in the form of a membrane orthin layer.

Device 160 in FIG. 9 is distinguished from device 100 in FIG. 6A in thatdevice 160 includes a vacuum reservoir 166, instead of a vacuum port, atthe terminus of delivery channel 170. In addition, sample inlet 162 indevice 160 is conveniently adapted to operate in conjunction with inletfitting 190, so that evacuation of the network and sample loading can beeffected from a single site with respect to the network.

Sample inlet 162 includes a hollow inlet cylinder 176 having an openproximal end 177, which connects to network 164, and an open, distal end178. Cylinder 176 further includes an opening 179 located near theterminus of the distal end.

Inlet fitting 190 includes an inlet cap structure 200 and a portstructure 210 appended thereto. Cap structure 200 defines a hollowcylinder 202 having an open, proximal end 204 and a closed, distal end206. The inner diameter of cylinder 202 is dimensioned to form avacuum-tight seal when placed over inlet 162. Port structure 210 definesa vacuum port 212 and a sample port 214. Ports 212 and 214 communicatewith cylinder 202 via openings 216 and 218, respectively, which areformed in the side of cylinder 202. Fitting 190 additionally includesguide structure 220 for receiving the adjacent edge of panel 174, toorient and guide fitting 190 when fitting 190 is fitted over and slidedalong inlet 162.

Exemplary dimensions of a device which has been prepared in accordancewith FIG. 9 are the following: detection chambers 168, 1.2 mm×1.2mm×0.75 mm; delivery channel 170, 0.25 mm×0.25 mm (width×depth);dead-end fluid connection 172, 0.25 mm×0.25 mm (width×depth); externaldimensions: 22 cm×15 cm×1 mm (dimensions of network-defining portion,excluding inlet 162 and panel 174). Preferably, the detection chambersin the microdevice of the invention have volumes less than 10 μL, lessthan 2 μL, and most preferably less than or equal to 1 μL.

Device 160 may be prepared under ordinary atmospheric conditions bybonding a polymeric, adhesive-backed substrate layer 180 to thecorresponding surface of layer 161, to form a sealed network. Inletfitting 190 is fitted onto cylinder 176 of inlet 162, such that openings179 and 216 are aligned with each other. Vacuum port 212 is connected toa vacuum line, and the interior of the network is evacuated for aselected time. The network may be alternately flushed with a selectedgas, such as carbon dioxide, and vacuum, as discussed above. Duringevacuation, sample port 214 is loaded with, or placed in fluidcommunication with, the fluid sample. Preferably, the sample port isfilled so that there is no air between the sample in the sample port andopening 218. Once the network has been evacuated (usually completewithin a few seconds), fitting 190 is lowered further towards layer 161until opening 179 is aligned with sample opening 218, bringing theinterior of the network in fluid communication with the sample. Thesample fills the chambers rapidly, typically in less than half a second.The detection chambers are filled to a volume-percent of greater than95%. Excess sample and residual gas collects in reservoir 166.

Once the chambers have filled with sample, fitting 190 is loweredfurther toward layer 161 in order to seal inlet 162, thereby sealing theinterior of the network from the outside atmosphere. The sample isallowed to react with the detection reagents in the detection chambers,during or after which the optical signals produced in the chambers aredetected.

C. Detection Reagents

The detection chamber(s) of the device may be pre-loaded with detectionreagents which are specific for the selected analytes of interest. Thedetection reagents are designed to produce an optically detectablesignal via any of the optical methods noted in Section II below.

It will be appreciated that although the reagents in each detectionchamber must contain substances specific for the analyte(s) to bedetected in the particular chamber, other reagents necessary to producethe optical signal for detection may be added to the sample prior toloading, or may be placed at locations elsewhere in the network formixing with the sample. Whether particular assay components are includedin the detection chambers or elsewhere will depend on the nature of theparticular assay, and on whether a given component is stable to drying.In general, it is preferred that as many of the detection reagents aspossible are pre-loaded in the detection chambers during manufacture ofthe device, in order to enhance assay uniformity and minimize the assaysteps conducted by the end-user.

The analyte to be detected may be any substance whose presence, absence,or amount is desireable to be determined. The detection means caninclude any reagent or combination of reagents suitable to detect ormeasure the analyte(s) of interest. It will be appreciated that morethan one analyte can be tested for in a single detection chamber, ifdesired.

In one embodiment, the analytes are selected-sequence polynucleotides,such as DNA or RNA, and the analyte-specific reagents includesequence-selective reagents for detecting the polynucleotides. Thesequence-selective reagents include at least one binding polymer whichis effective to selectively bind to a target polynucleotide having adefined sequence.

The binding polymer can be a conventional polynucleotide, such as DNA orRNA, or any suitable analog thereof which has the requisite sequenceselectivity. For example, binding polymers which are analogs ofpolynucleotides, such as deoxynucleotides with thiophosphodiesterlinkages, and which are capable of base-specific binding tosingle-stranded or double-stranded target polynucleotides may be used.Polynucleotide analogs containing uncharged, but stereoisomericmethylphosphonate linkages between the deoxyribonucleoside subunits havebeen reported (Miller, 1979, 1980, 1990, Murakami, Blake, 1985a, 1985b).A variety of analogous uncharged phosphoramidate-linked oligonucleotideanalogs have also been reported (Froehler). Also, deoxyribonucleosideanalogs having achiral and uncharged intersubunit linkages (Stirchak)and uncharged morpholino-based polymers having achiral intersubunitlinkages have been reported (U.S. Pat. No. 5,034,506). Binding polymersknown generally as peptide nucleic acids may also be used (Buchardt,1992). The binding polymers may be designed for sequence specificbinding to a single-stranded target molecule through Watson-Crick basepairing, or sequence-specific binding to a double-stranded targetpolynucleotide through Hoogstein binding sites in the major groove ofduplex nucleic acid (Kornberg). A variety of other suitablepolynucleotide analogs are also known.

The binding polymers for detecting polynucleotides are typically 10-30nucleotides in length, with the exact length depending on therequirements of the assay, although longer or shorter lengths are alsocontemplated.

In one embodiment, the analyte-specific reagents include anoligonucleotide primer pair suitable for amplifying, by polymerase chainreaction, a target polynucleotide region of the selected analyte whichis flanked by 3′-sequences complementary to the primer pair. Inpracticing this embodiment, the primer pair is reacted with the targetpolynucleotide under hybridization conditions which favor annealing ofthe primers to complementary regions of opposite strands in the target.The reaction mixture is then thermal cycled through several, andtypically about 20-40, rounds of primer extension, denaturation, andprimer/target sequence annealing, according to well-known polymerasechain reaction (PCR) methods (Mullis, Saiki).

Typically, both primers for each primer pair are pre-loaded in each ofthe respective detection chambers, along with the standard nucleotidetriphosphates, or analogs thereof, for primer extension (e.g., ATP, CTP,GTP, and TTP), and any other appropriate reagents, such as MgCl₂ orMnCl₂. A thermally stable DNA polymerase, such as Taq, Vent, or thelike, may also be pre-loaded in the chambers, or may be mixed with thesample prior to sample loading. Other reagents may be included in thedetection chambers or elsewhere as appropriate. Alternatively, thedetection chambers may be loaded with one primer from each primer pair,and the other primer (e.g., a primer common to all of detectionchambers) may be provided in the sample or elsewhere.

If the target polynucleotides are single-stranded, such assingle-stranded DNA or RNA, the sample is preferably pre-treated with aDNA- or RNA-polymerase prior to sample loading, to form double-strandedpolynucleotides for subsequent amplification.

The presence and/or amount of target polynucleotide in a detectionchamber, as indicated by successful amplification, is detected by anysuitable means. For example, amplified sequences may be detected indouble-stranded form by including an intercalating or cross-linking dye,such as ethidium bromide, acridine orange, or an oxazole derivative, forexample, which exhibits a fluorescence increase or decrease upon bindingto double-stranded nucleic acids (Sambrook, 1989; Ausubel; Higuchi,1992, 1993; Ishiguro, 1995).

The level of amplification can also be measured by fluorescencedetection using a fluorescently labeled oligonucleotide, such asdisclosed in Lee et al. (1993) and Livak et al. (1995). In thisembodiment, the detection reagents include a sequence-selective primerpair as in the more general PCR method above, and in addition, asequence-selective oligonucleotide (FQ-oligo) containing afluorescer-quencher pair. The primers in the primer pair arecomplementary to 3′-regions in opposing strands of the target analytesegment which flank the region which is to be amplified. The FQ-oligo isselected to be capable of hybridizing selectively to the analyte segmentin a region downstream of one of the primers and is located within theregion to be amplified.

The fluorescer-quencher pair includes a fluorescer dye and a quencherdye which are spaced from each other on the oligonucleotide so that thequencher dye is able to significantly quench light emitted by thefluorescer at a selected wavelength, while the quencher and fluorescerare both bound to the oligonucleotide. The FQ-oligo preferably includesa 3′-phosphate or other blocking group to prevent terminal extension ofthe 3′-end of the oligo.

The fluorescer and quencher dyes may be selected from any dyecombination having the proper overlap of emission (for the fluorescer)and absorptive (for the quencher) wavelengths while also permittingenzymatic cleavage of the FQ-oligo by the polymerase when the oligo ishybridized to the target. Suitable dyes, such as rhodamine andfluorscein derivatives, and methods of attaching them, are well knownand are described, for example, in Menchen et al. (1993, 1994), Bergotet al. (1991), and Fung et al. (1987).

The fluorescer and quencher dyes are spaced close enough together toensure adequate quenching of the fluorescer, while also being far enoughapart to ensure that the polymerase is able to cleave the FQ-oligo at asite between the fluorescer and quencher. Generally, spacing of about 5to about 30 bases is suitable, as generally described in Livak et al.(1995). Preferably, the fluorescer in the FQ-oligo is covalently linkedto a nucleotide base which is 5′ with respect to the quencher.

In practicing this approach, the primer pair and FQ-oligo are reactedwith a target polynucleotide (double-stranded for this example) underconditions effective to allow sequence-selective hybridization to theappropriate complementary regions in the target. The primers areeffective to initiate extension of the primers via DNA polymeraseactivity. When the polymerase encounters the FQ-probe downstream of thecorresponding primer, the polymerase cleaves the FQ-probe so that thefluorescer is no longer held in proximity to the quencher. Thefluorescence signal from the released fluorescer therefore increases,indicating that the target sequence is present.

One advantage of this embodiment is that only a small proportion of theFQ-probe need be cleaved in order for a measurable signal to beproduced. In a further embodiment, the detection reagents may includetwo or more FQ-oligos having distinguishable fluorescer dyes attached,and which are complementary for different-sequence regions which may bepresent in the amplified region, e.g., due to heterozygosity (Lee,1993).

In another embodiment, the detection reagents include first and secondoligonucleotides effective to bind selectively to adjacent, contiguousregions of a target sequence in the selected analyte, and which may beligated covalently by a ligase enzyme or by chemical means (Whiteley,1989; Landegren, 1988) (oligonucleotide ligation assay, OLA). In thisapproach, the two oligonucleotides (oligos) are reacted with the targetpolynucleotide under conditions effective to ensure specifichybridization of the oligonucleotides to their target sequences. Whenthe oligonucleotides have base-paired with their target sequences, suchthat confronting end subunits in the oligos are basepaired withimmediately contiguous bases in the target, the two oligos can be joinedby ligation, e.g., by treatment with ligase. After the ligation step,the detection wells are heated to dissociate unligated probes, and thepresence of ligated, target-bound probe is detected by reaction with anintercalating dye or by other means.

The oligos for OLA may also be designed so as to bring together afluorescer-quencher pair, as discussed above, leading to a decrease in afluorescence signal when the analyte sequence is present.

In the above OLA ligation method, the concentration of a target regionfrom an analyte polynucleotide can be increased, if necessary, byamplification with repeated hybridization and ligation steps. Simpleadditive amplification can be achieved using the analyte polynucleotideas a target and repeating denaturation, annealing, and ligation stepsuntil a desired concentration of the ligated product is achieved.

Alternatively, the ligated product formed by hybridization and ligationcan be amplified by ligase chain reaction (LCR), according to publishedmethods (Winn-Deen). In this approach, two sets of sequence-specificoligos are employed for each target region of a double-stranded nucleicacid. One probe set includes first and second oligonucleotides designedfor sequence-specific binding to adjacent, contiguous regions of atarget sequence in a first strand in the target. The second pair ofoligonucleotides are effective to bind (hybridize) to adjacent,contiguous regions of the target sequence on the opposite strand in thetarget. With continued cycles of denaturation, reannealing and ligationin the presence of the two complementary oligo sets, the target sequenceis amplified exponentially, allowing small amounts of target to bedetected and/or amplified.

In a further modification, the oligos for OLA or LCR assay bind toadjacent regions in a target polynucleotide which are separated by oneor more intervening bases, and ligation is effected by reaction with (i)a DNA polymerase, to fill in the intervening single stranded region withcomplementary nucleotides, and (ii) a ligase enzyme to covalently linkthe resultant bound oligonucleotides (Segev, 1992, 1994).

In another embodiment, the target sequences can be detected on the basisof a hybridization-fluorescence assay (Lee et al., 1993). The detectionreagents include a sequence-selective binding polymer (FQ-oligo)containing a fluorescer-quencher pair, as discussed above, in which thefluorescence emission of the fluorescer dye is substantially quenched bythe quencher when the FQ-oligo is free in solution (i.e., not hybridizedto a complementary sequence).

Hybridization of the FQ-oligo to a complementary sequence in the targetto form a double-stranded complex is effective to perturb (e.g.,increase) the fluorescence signal of the fluorescer, indicating that thetarget is present in the sample. In another embodiment, the bindingpolymer contains only a fluorescer dye (but not a quencher dye) whosefluorescence signal either decreases or increases upon hybridization tothe target, to produce a detectable signal.

It will be appreciated that since the selected analytes in the samplewill usually be tested for under generally uniform temperature andpressure conditions within the device, the detection reagents in thevarious detection chambers should have substantially the same reactionkinetics. This can generally be accomplished using oligonucleotides andprimers having similar or identical melting curves, which can bedetermined by empirical or experimental methods as are known in the art.

In another embodiment, the analyte is an antigen, and theanalyte-specific reagents in each detection chamber include an antibodyspecific for a selected analyte-antigen. Detection may be byfluorescence detection, agglutination, or other homogeneous assayformat. As used herein, “antibody” is intended to refer to a monoclonalor polyclonal antibody, an Fc portion of an antibody, or any other kindof binding partner having an equivalent function.

For fluorescence detection, the antibody may be labeled with afluorescer compound such that specific binding of the antibody to theanalyte is effective to produce a detectable increase or decrease in thecompound's fluorescence, to produce a detectable signal (non-competitiveformat). In an alternative embodiment (competitive format), thedetection means includes (i) an unlabeled, analyte-specific antibody,and (ii) a fluorescer-labeled ligand which is effective to compete withthe analyte for specifically binding to the antibody. Binding of theligand to the antibody is effective to increase or decrease thefluorescence signal of the attached fluorescer. Accordingly, themeasured signal will depend on the amount of ligand that is displaced byanalyte from the sample. Exemplary fluorescence assay formats which maybe adapted for the present invention can be found in Ullman (1979, 1981)and Yoshida (1980), for example.

In a related embodiment, when the analyte is an antibody, theanalyte-specific detection reagents include an antigen for reacting witha selected analyte antibody which may be present in the sample. Thereagents may be adapted for a competitive or non-competitive typeformat, analogous to the formats discussed above. Alternatively, theanalyte-specific reagents include a mono- or polyvalent antigen havingone or more copies of an epitope which is specifically bound by theantibody-analyte, to promote an agglutination reaction which providesthe detection signal.

In yet another embodiment, the selected analytes are enzymes, and thedetection reagents include enzyme-substrate molecules which are designedto react with specific analyte enzymes in the sample, based on thesubstrate specificities of the enzymes. Accordingly, detection chambersin the device each contain a different substrate or substratecombination, for which the analyte enzyme(s) may be specific. Thisembodiment is useful for detecting or measuring one or more enzymeswhich may be present in the sample, or for probing the substratespecificity of a selected enzyme. Particularly preferred detectionreagents include chromogenic substrates such as NAD/NADH, FAD/FADH, andvarious other reducing dyes, for example, useful for assayinghydrogenases, oxidases, and enzymes that generate products which can beassayed by hydrogenases and oxidases. For esterase or hydrolase (e.g.,glycosidase) detection, chromogenic moieties such as nitrophenol may beused, for example.

In another embodiment, the analytes are drug candidates, and thedetection reagents include a suitable drug target or an equivalentthereof, to test for binding of the drug candidate to the target. Itwill be appreciated that this concept can be generalized to encompassscreening for substances that interact with or bind to one or moreselected target substances. For example, the assay device can be used totest for agonists or antagonists of a selected receptor protein, such asthe acetylcholine receptor. In a further embodiment, the assay devicecan be used to screen for substrates, activators, or inhibitors of oneor more selected enzymes. The assay may also be adapted to measuredose-response curves for analytes binding to selected targets.

The sample or detection reagents may also include a carrier protein,such as bovine serum albumin (BSA) to reduce non-specific binding ofassay components to the walls of the detection chambers.

The analyte-specific detection reagents are preferably dispensed intothe detection chambers robotically using a dispensing system designed todeliver small volumes of liquid solutions (e.g., 0.1 to 1 μL). Thesystem is supplied with separate analyte-specific detection reagentswhich are dispensed to pre-selected detection chambers withoutcross-contamination.

A reagent loading device that has been prepared in accordance with theinvention includes a dispensing robot (Asymtek Automove 402) coupled toa plurality of drop-on-demand ink-jet printing heads. The robot includesan X,Y-axis work table (12 inch×12 inch) having a lateral resolution of0.001 inch, a lateral velocity of 0-20 inch/sec, a Z-axis resolution of0.001 inch, and a Z-axis velocity of 0-8 inch/sec. The robot optionallyincludes a tip locator, offset camera, strobe drop camera, on-axiscamera, and/or gravimetric drop calibration. The printing heads are of adrop-on-demand, piezo-electric type, having wetted surfaces usuallyselected from glass, Teflon, and polypropylene. The minimum drop volumeis 25 nL, and the maximum flow is 1 μL/min.

Reagent loading is preferably accomplished under carefully-controlledsterile conditions using one or more dedicated dispensing robots. Afterapplication, the reagents are allowed to dry in the chambers until mostor all of the solvent has evaporated. Drying may be accelerated bybaking or reduced pressure as appropriate. The detection chambers arethen sealed by bonding the chamber containing substrate layer with anappropriate cover layer, and the device is ready for use.

III. Signal Detection and Analysis

The signal produced by reaction of the analyte-specific reagents withthe sample is measured by any suitable detection means, includingoptical and non-optical methods.

Where the signal is detected optically, detection may be accomplishedusing any optical detector that is compatible with the spectroscopicproperties of the signal. The assay may involve an increase in anoptical signal or a decrease. The optical signal may be based on any ofa variety of optical principals, including fluorescence,chemiluminescence, light absorbance, circular dichroism, opticalrotation, Raman scattering, radioactivity, and light scattering.Preferably, the optical signal is based on fluorescence,chemiluminescence, or light absorbance.

In general, the optical signal to be detected will involve absorbance oremission of light having a wavelength between about 180 nm (ultraviolet)and about 50 μm (far infrared). More typically, the wavelength isbetween about 200 nm (ultraviolet) and about 800 nm (near infrared). Avariety of detection apparatus for measuring light having suchwavelengths are well known in the art, and will typically involve theuse of light filters, photomultipliers, diode-based detectors, and/orcharge-coupled detectors (CCD), for example.

The optical signals produced in the individual detection chambers may bemeasured sequentially by iteratively scanning the chambers one at a timeor in small groups, or may be measured simultaneously using a detectorwhich interrogates all of the detection chambers continuously or atshort time intervals. Preferably, the signals are recorded with the aidof a computer capable of displaying instantaneously (in real-time) thesignal level in each of the detection chambers, and also storing thetime courses of the signals for later analysis.

The optical signal in each chamber may be based on detection of lighthaving one or more selected wavelengths with defined band-widths (e.g.,500 nm±5 μm). Alternatively, the optical signal may be based on theshape or profile of emitted or absorbed light in a selected wave-lengthrange. Preferably, the optical signal will involve measurement of lighthaving at least two distinctive wavelengths in order to include aninternal control. For example, a first wavelength is used to measure theanalyte, and a second wavelength is used to verify that the chamber isnot empty or to verify that a selected reagent or calibration standardis present in the detection chamber. An aberration or absence of thesignal for the second wavelength is an indication that the chamber maybe empty, that the sample was improperly prepared, or that the detectionreagents are defective.

In studies conducted in support of the invention, a detection assemblywas prepared for fluorescence detection of target polynucleotides in asample using a device in accordance with the invention. The assemblyincludes a translation stage for positioning the test device. The testdevice includes a 7×7 array of addressable detection chambers containingfluorescent detection reagents. The detector in the assembly consists ofa tungsten bulb (or quartz halogen bulb, 75 W) illumination source, aCCD camera, and appropriate focusing/collection optics. The illuminationsource is positioned so as to illuminate the device diagonally fromabove (e.g., at an inclination angle of 45 degrees with respect to theilluminated surface). The optics include two lenses separated by anemission filter. The first lens collimates the incoming image for theemission filter, and the second lens which re-images the filtered beamonto the CCD. The test device is placed at the focal point of the firstlens.

The CCD is a thermoelectrically cooled, instrumentation-gradefront-illuminated CCD (Princeton Instruments TEA/CCD-512TK/1). Thedetection plate of the CCD has a 512×512 array of 27 μm square pixelswhich covers the entire overhead cross-section of the test device. Thecamera head is controlled by a controller (Princeton Instruments ST-135)which communicates with a computer (Quadra 650, Apple Computers) forcollecting and processing the signal data. The system is capable ofon-chip binning of the pixels. For detection chambers having an overheadcross-section of 1 mm×1 mm, bins having a size of 2×2 pixels aresuitable. More generally, the bin size is selected on the basis of thetotal processing time that will be required, the sizes and number ofdetection chambers, sensitivity, and signal noise.

The computer in the assembly includes signal-processing software forselecting an appropriate sub-region in each detection chamber from whichthe signal is measured. Such sub-regions are selected for uniformity ofincoming light, to ensure that edge regions are excluded. The signalimage of the device is recorded and stored at selected intervals,according to the requirements of the assay. Preferably, the signal foreach detection chamber is recorded as an average signal per bin for theselected sub-region in each chamber, since the size of the selectedsub-region in each chamber will usually differ from chamber to chamber.

The detector optics may further be adapted to include a filter wheel fordetecting fluorescence at 2 or more wavelengths.

As discussed above, the temperature of the detection chambers may becontrolled, if appropriate, by any of a number of suitable methods. Inthe detection assembly that was prepared in accordance with theinvention, the heating means is external to the test device (off-chipheating), and includes a temperature controller (Marlow Industries modelSE 5020) equipped with a peltier device (ITI Ferro Tec model 6300)having a ramp rate of about ±4 E/sec in the range of 55 EC to 95 EC. Foron-chip heating, where the device includes resistive tracings (or acomparable equivalent) for heating the chambers, the assembly can bemodified to provide one or two zones of resistance heating capable ofestablishing a maximum power dissipation of 25 W over a 200 mm² area;this mode can provide a ramp rate of ±10 E/sec during transition from 55EC to 95 EC.

The above described structure (Section IIB) for detecting ananalyte-related signal in each chamber, including the optical windowassociated with that chamber, is also referred to herein collectively asdetection means for detecting such signal.

Another type of detection means is a biosensor device capable ofdetecting the reaction of an analyte with an analyte-specific reagent ineach chamber. Amperometric biosensors suitable for use in the inventionoperate on a variety of principles. In one, the analyte being measuredis itself capable of interacting with an analyte-specific reagent togenerate an electrochemical species, i.e., a species capable of functionas an electron donor or acceptor when in contact with an electrode. Asan example, reaction of the analyte cholesterol with the reagentcholesterol oxidase generates the electrochemical species H₂O₂ which, incontact with an electrode, produces a measurable current in a circuitcontaining the electrode.

The analyte-specific reagent may be localized on a film separated fromthe electrode surface by a permselective layer that is selectivelypermeable to the electrochemical species (and other small components inthe sample). When sample fluid is added to the biosensor, reaction ofthe analyte with the corresponding reagent produces an electrochemicalspecies whose presence and amount are quantitated by current measurementthrough the electrode.

Alternatively, the analyte-specific reagent may be a receptor which isspecific for the analyte. Initially, the receptor sites are filled withan analyte-enzyme conjugate. In the presence of analyte, the conjugatesare displaced from the receptor, and are then free to migrate topositions close to the electrode, for production of transientelectrochemical species (such as H₂O₂ in the presence of catalase) inthe vicinity of the electrode.

Another general type of biosensor employs a lipid bilayer membrane as agate for electrochemical species interposed between a sample-fluidchamber and an electrode. The bilayer is provided with ion-channelproteins which function as ion gates that can be opened by analytebinding to the proteins. Thus, binding of analyte to the channelproteins (which serve as the analyte-specific reagent) leads to ion flowacross the membrane and detectable signal at the electrode.

Thin-film biosensors of the type mentioned above may be formed in amicrochip or small-substrate format by photolithographic methods, suchas described in U.S. Pat. Nos. 5,391,250, 5,212,050, 5,200,051, and4,975,175. As applied to the present invention, the chamber walls in thesubstrate may serve as the substrate for deposition of the requiredelectrode and film layers. In addition to these layers, suitableconductive connectors connecting the electrodes to electrical leads arealso laid down.

In a typical device, each chamber contains a biosensor for a givenanalyte. When sample is introduced into the device, the multiple sampleanalytes are then separately measured in the chambers, with the resultsbeing reported to a processing unit to which the device is electricallyconnected.

IV. Assay Method

In another aspect, the invention includes a method for detecting orquantitating a plurality of analytes in a liquid sample. In the method,there is provided a device of the type described above, wherein theinterior of the network is placed under vacuum. A liquid sample is thenapplied to the inlet, and the sample is allowed to be drawn into thesample-distribution network by vacuum action, delivering sample to thedetection chambers. The delivered sample is allowed to react with theanalyte-specific reagent in each detection chamber under conditionseffective to produce a detectable signal when the selected analyte ispresent in the sample. The reaction chambers are inspected or analyzedto determine the presence and/or amount of the selected analytes in thesample.

The sample tested may be from any source which can be dissolved orextracted into a liquid that is compatible with the device, and whichmay potentially contain one or more of the analytes of interest. Forexample, the sample may be a biological fluid such as blood, serum,plasma, urine, sweat, tear fluid, semen, saliva, cerebral spinal fluid,or a purified or modified derivative thereof. The sample may also beobtained from a plant, animal tissue, cellular lysate, cell culture,microbial sample, or soil sample, for example. The sample may bepurified or pre-treated if necessary before testing, to removesubstances that might otherwise interfere with analyte detection.Typically, the sample fluid will be an aqueous solution, particularlyfor polar analytes such as polypeptides, polynucleotides, and salts, forexample. The solution may include surfactants or detergents to improveanalytes solubility. For non-polar and hydrophobic analytes, organicsolvents may be more suitable.

As discussed above, the device may be manufactured and sold in a formwherein the sample-distribution network is under vacuum, so that thedevice is ready to load by the end-user. Alternatively, evacuation ofthe network is conducted by the user, through a vacuum port or via thesample inlet itself.

Prior to sample loading, any gas in the network may be replaced withanother gas, according to the requirements of the assay. In a preferredembodiment, the residual gas is replaced with carbon dioxide, so thatany gas bubbles that appear in the network after sample loading arequickly dissolved by the sample fluid, particularly if the sample is anaqueous solution.

It will be appreciated that when the device includes a vacuum portdownstream of the detection chambers, the sample delivery channels inthe device may be cleared of sample after the detection chambers havebeen filled, to further isolate the detection chambers from each other.The invention also contemplates filling the delivery channels with anadditional fluid, such as a mineral oil or a viscous polymer solutioncontaining agarose or other viscous material (e.g., see Dubrow, U.S.Pat. No. 5,164,055, and Menchen et al., U.S. Pat. No. 5,290,418), tosegregate the chambers from each other, or with a reagent-containingsolution which facilitates the assay.

In a particularly advantageous embodiment of the invention, alarge-volume syringe can be used to generate a vacuum inside the sampledistribution network of the device prior to loading. By “large-volume”is meant that the volume of the syringe is greater than the totalinternal volume of the device (i.e., of the sample distributionnetwork). Preferably, the volume of the syringe is at least 20-foldgreater than the interior volume of the device. With reference to thedevice in FIG. 9, the inlet-tip of the syringe is connected to vacuumport 212. When opening 216 is aligned with opening 179, the syringe isused to draw air from the interior of the device, thereby lowering theinternal pressure. For example, if a syringe with a volume of 50 mL isused, and the internal volume of the device is 100 μL, the pressure inthe sample distribution network can be reduced by a factor of 500 (=0.1mL/50 mL). Thus, an initial internal pressure of 760 torr can be reducedto less than 2 torr. With reference to the device in FIG. 6A, thesyringe can be connected to fitting 106 or 102 using appropriateconnections, to withdraw air from the distribution network.

Accordingly, the present invention includes a kit comprising (i) adevice as described above and (ii) a syringe for drawing air from theinterior of the device. The invention also includes a method of usingthe kit to detect one or more analytes in a sample, as described above.It will be appreciated that using a syringe greatly simplifies the stepof creating a vacuum inside the device, so that the device can be usedquickly or immediately without needing a mechanical vacuum pump.

V. Utility

The present invention can be used in a wide variety of applications. Theinvention can be used for medical or veterinary purposes, such asdetecting pathogens, diagnosing or monitoring disease, geneticscreening, determining antibody or antigen titers, detecting andmonitoring changes in health, and monitoring drug therapy. The inventionis also useful in a wide variety of forensic, environmental, andindustrial applications, including screening drug candidates foractivity.

More generally, the present invention provides a convenient method forsimultaneous assay of multiple analytes in a sample. The invention ishighly flexible in its applications, being adaptable to analysis of awide variety of analytes and sample materials. By providingpre-dispensed, analyte-specific reagents in separate detection chambers,the invention eliminates the need for complicated and time-consumingreagent preparation.

Practice of the invention is further simplified since the detectionchambers can be loaded via a single sample inlet. The use of uniformlysized detection chambers renders the device self-metering, in that aprecise volume of sample is delivered to each chamber. Thus, theprecision, accuracy and reproducibility of the assay are all enhanced,since the quantities and compositions of the analyte-specific reagents,the quantity of sample in the chambers, and the reaction conditions canbe carefully controlled. Moreover, very small volumes of sample arerequired since the dimensions of the sample-distribution network in thedevice can be very small.

The device may be formed from a wide variety of materials, allowing thecomposition of the device to be adapted to the particular reagents andconditions in the assay. Inasmuch as the device requires no movingparts, and can be relatively small in size (typically having dimensionon the order of millimeters to centimeters), manufacture of the deviceis simplified and costs are reduced.

The features and advantages of the invention may be further understoodfrom the following example, which is not intended in any way to limitthe scope of the invention.

EXAMPLE

The following study was performed using a polycarbonate microdevicesubstantially as shown in FIG. 9, to demonstrate detection of a humanβ-actin gene by PCR (polymerase chain reaction). The assay componentsfor PCR detection were obtained from PE Applied Biosystems (Foster City,Calif., β-actin kit, part #N808-0230). The kit components included thefollowing stock solutions:

β-actin forward primer:

-   3 μM primer in 10 mM Tris-HCl, pH 8.0 (at room temperature), 1 mM    EDTA    β-actin reverse primer:-   3 μM primer in 10 mM Tris-HCl, pH 8.0 (at room temperature), 1 mM    EDTA    β-actin probe:-   2 μM TAMRA-labeled probe in 10 mM Tris-HCl, pH 8.0 (at room    temperature), 1 mM EDTA    DNA sample:-   370 μg/mL human genomic DNA in 10 mM Tris-HCl, pH 8.0 (at room    temperature), 1 mM EDTA (from Coriell Cell Repositories, Camden,    N.J.)    dNTPs:-   20 mM dNTP (1 tube each for dUTP, dATP, dCTP and dGTP) in autoclaved    deionized ultrafiltered water, titrated to pH 7.0 with NaOH    DNA polymerase:-   “AMPLITAQ GOLD” DNA polymerase at 5 U/μL, from PE Applied    Biosystems, part #N808-0240 (PE Applied Biosystems “AMPLITAQ GOLD”    Product Brochure, 1996)    “AMPERASE” UNG:-   uracil-N-glycosylase at 1 U/μL, from PE Applied Biosystems, part    #N808-0096    10× “TAQMAN” buffer A:-   500 mM KCl, 100 mM Tris-HCl, 0.1 M EDTA, 600 nM Passive Reference 1    (ROX), pH 8.3 at room temperature, autoclaved    MgCl₂:-   20 mM MgCl₂ in autoclaved deionized ultrafiltered water

A description of the sequences of the forward primer, the reverseprimer, and the TAMRA-labeled probe can be found in PE AppliedBiosystems “TAQMAN” PCR Reagent Protocol (1996), which also describesthe general steps of the “TAQMAN” assay technique. The forward andreverse primers were effective to produce a 297 basepair PCR product.

A flat substrate layer 180 and a substrate layer 161 were formed frompolycarbonate by standard injection-molding methods (substrate layer161) or from sheet stock (layer 180). The volume of each detectionchamber was 1 μL.

Detection chambers were loaded with different amounts of forward primer,reverse primer, and fluorescent probe as follows. To a polypropylenetube was added 0.5 mL each of β-actin forward primer solution, reverseprimer solution, and fluorescent probe, to give a final primer/probestock solution volume of 1.5 mL. This solution was then loaded intoalternating detection chambers in substrate layer 161 using arobotically controlled microsyringe. Specifically, alternate chamberswere loaded with either a 1×, 5× or 10× amount of primer/probe solution,with 1× (14 nL primer/probe stock solution) being equivalent to a finalconcentration in a detection chamber of 15 nM of each primer and 10 nMof fluorescent probe (after the dried chamber is subsequently filledwith sample), 5× (72 nL primer/probe stock solution) being equivalent toa final concentration of 75 nM of each primer and 50 nM of fluorescentprobe, and 10× (145 nL primer/probe stock solution) being equivalent toa final concentration of 150 nM of each primer and 100 nM of fluorescentprobe. The amounts of primer and probe in the loaded chamberscorresponded to 1/20, ¼, and ½ of the concentrations used under standardreaction conditions, for the 1×, 5×, and 10× chambers, respectively. Theloaded chambers produced a “checkerboard” pattern in substrate layer 161where each loaded chamber was separated by an intervening empty chamber.

After the loaded chambers were allowed to air-dry to dryness at roomtemperature, the loaded substrate layer (161) was joined to a flatsubstrate layer 180 by ultrasonic welding. Inlet fitting 190 was thenplaced over sample inlet 162, such that opening 179 was aligned withvacuum port opening 216. The sample distribution network 164 anddetection chambers 168 were evacuated via vacuum port 212, which wasconnected to a vacuum pump, to a final internal pressure ofapproximately 1 to 10 torr.

The PCR reaction mixture (sample), without primers and probe, wasprepared from the above stock solutions to give the following finalconcentrations in the sample:

-   -   10 mM Tris-HCl, pH 8.3    -   50 mM KCl    -   3.5 mM MgCl₂    -   400 μM dUTP    -   200 μM each dATP, dCTP, and dGTP    -   0.01 U/μL uracil-N-glycosylase    -   0.25 U/μL “AMPLITAQ GOLD” DNA polymerase    -   0.74 ng/μL human genomic DNA template

For loading of sample into the microdevice, a micropipette loaded withthe above sample solution was placed in sample port 214 so as tominimize the deadvolume occupied by air at the tip of the pipette. Inletfitting 190 was then pressed down further to align opening 179 withopening 218, so that the sample was drawn from port 214 into thedetection chambers by vacuum action. Filling of the chambers wascomplete in less than a second.

The microdevice was then clamped to a peltier device (20 mm×20 mm) gluedto an aluminum heat sink. Cycling was controlled using a Marlowtemperature controller (Marlow Industries Inc., Dallas, Tex., Model No.SE 5020). A thermistor was attached to the peltier device to providetemperature feedback (Marlow part No. 217-2228-006). The microdevice wasthermocycled as follows:

-   -   1) precycle: 50 EC for 2 minutes; 95 EC for 10 minutes;    -   2) 40 cycles: 95 EC for 15 seconds, 60 EC for 1 minute;    -   3) hold at 72 EC.

Signal detection was accomplished using a fluorescence detectioninstrument consisting of a tungsten bulb for illumination and a CCDcamera and 4-color filter wheel for detection. Images of all detectionchambers (wells) were taken at the end of each thermocycle (during the60 EC step) at several wavelengths in order to monitor the increase ofthe reporter's fluorescence. Interfering fluorescence fluctuations werenormalized by dividing the emission intensity of the reporter dye by theemission intensity of the passive reference (ROX dye) for a givenchamber. The excitation wavelength was 488 nm. The reporter intensitywas measured at 518 nm, and the passive reference intensity was measuredat 602 nm.

Results. Positive fluorescent signals were detected in all chambers thathad been loaded with the β-actin primers and fluorescent probe at the 5×and 10 concentrations. Little or no signal was detected for chambersloaded at the 1× concentration. No detectable signal was detected abovebackground for the chambers which did not contain β-actin primer andprobe, indicating that there was no cross-contamination betweendetection chambers after 40 heat/cool cycles.

The highest final fluorescence signals were obtained in detectionchambers loaded with a 10× amount of primers and probe, with detectablesignals appearing after about 23 cycles. The 5× chambers also showeddetectable signals after cycle 23, but the final fluorescence signal wasnot as high as that for the 10× wells (due to lower probeconcentration). Thus, the 3-actin gene was readily detected using primerand probe concentrations equal to ¼ and ½ of those used under ordinaryconditions. The results also show that the preloaded primers and probeswere successfully dissolved in the sample after sample loading.

Although the invention has been described by way of illustration andexample for purposes of clarity and understanding, it will beappreciated that various modifications can be made without departingfrom the invention. All references cited above are incorporated hereinby reference.

1. A device comprising: a substrate; a sample-distribution networkdefined by the substrate and comprising (i) a sample inlet, (ii) two ormore detection chambers, (iii) at least one sample delivery channel influid communication with the sample inlet, and (iv) a plurality ofdead-end fluid connections, each of the dead-end fluid connectionsproviding a dead-end communication between a respective one of the twoor more detection chambers and the at least one sample delivery channel;and detection reagents in the detection chambers; wherein the substratecomprises two or more layers, a high thermal conductivity metal surface,and a plastic polymer coating on the high thermal conductivity metalsurface, and each of the two or more detection chambers is defined bythe plastic polymer coating such that the plastic polymer coatingseparates the high thermal conductivity metal surface from the detectionchamber.
 2. The device of claim 1, wherein the high thermal conductivitymetal surface comprises copper.
 3. The device of claim 1, wherein thehigh thermal conductivity metal surface comprises aluminum.
 4. Thedevice of claim 1, further comprising an optically clear cover layerdisposed over the sample distribution network.
 5. The device of claim 1,wherein at least one of the two or more detection chambers comprises adried detection reagent.
 6. The device of claim 1, wherein the substratedefines at least two such sample-distribution networks.
 7. The device ofclaim 1, further comprising means for modulating the temperature of thedetection chambers, in the substrate.
 8. The device of claim 1, whereinthe two or more layers are laminated together.
 9. A system comprisingthe device of claim 1 and an external temperature controller.